Investigation of prevalence and phylogenetic classification of Microsporidia MB and insecticide target site insensitivity resistance mutations in Anopheles gambiae s.l. and Anopheles funestus mosquitoes from Busia, Kenya.

Background: Microsporidia MB, a Plasmodium-transmission-impairing symbiont in Anopheles arabiensis, has malaria control potential. This study assessed its prevalence and phylogeny in An. gambiae s.l. and An. funestus in Busia, Kenya and investigated the influence of environmental factors on its occurrence. Additionally, the prevalence of key insecticide resistance mutations in these mosquito populations was determined.

Methods: Mosquito larvae and adults were collected from three sub-counties in Busia County, Kenya and identified based on morphological characteristics. PCR was used to determine Anopheles species distribution and Microsporidia MB prevalence following DNA extraction from the samples. Insecticide resistance target-site mutations were identified using TaqMan genotyping in a subset of the mosquito samples. Multivariable logistic regression models were used to assess associations of Microsporidia MB infection and ecological factors. Microsporidia MB-positive samples were whole-genome sequenced and phylogenetically analysed.

Results: Overall, An. gambiae s.l. (including An. gambiae s.s. and possibly An. coluzzii) comprised 57.3% of samples analysed while An. funestus comprised 25.7% and An. arabiensis 17% and their distribution varied significantly across the three sub-counties (Chi-square, χ² = 577.44, df = 4, p < 0.001). Microsporidia MB prevalence was low to moderate (0 to 6.4%) and highest in An. gambiae s.l. Anopheles gambiae s.l. showed significantly higher odds of infection compared to An. arabiensis (aOR = 5.94, 95% CI: 1.96-26.77, p = 0.006). Larvae reared to adults had significantly lower odds of infection than indoor-collected adults (aOR = 0.48, 95% CI: 0.26-0.86, p = 0.014). Insecticide resistance genotyping revealed high frequencies of kdr-East (94.7%) and kdr-West (60%) mutations in the Anopheles subset analysed, while Ace-1 and GSTe2 mutations were absent. Phylogenetic analysis placed Microsporidia MB isolated from Busia, within Clade IV, closely related to the originally sequenced Ahero reference, but still distinct from other microsporidian clades (I and III).

Conclusion: The present study highlights the occurrence of Microsporidia MB in multiple Anopheles vectors associated with malaria suggesting its broader potential as a vector control tool. The high prevalence of kdr mutations indicate a significant challenge to insecticide-based vector control in the region. Further investigation into the phenotypic expression of insecticide resistance in these populations is important. Results of the phylogenetic analysis suggest a common ancestry for Microsporidia MB isolates from Busia with the Ahero reference one, highlighting shared traits with potential for malaria control.